PEDV N interactiome _Label-Free.xlsx

Porcine epidemic diarrhea virus (PEDV) is the agent of PED which causes numerous economic losses for the swine industry each year. Till now, no effective vaccine or drugs are available to contain this disease. As a result, it is critical urgent to elucidate the PEDV interactome. The nucleocapsid (N) of PEDV plays a key role in viral replication. In this study, the N gene was cloned into pEGFP-C1 and the recombinant plasmid pEGFP-N was prepared. In order to obtain the interactome of N protein, the plasmids pEGFP-N and pEGFP-C1 were transfected in 293T and the cellular binding partners were pulled down using the GFP-trap. These proteins were identified by label-free MS. The results showed that approximately 1200 cellular proteins were initially identified and quantified, which represented both specific and nonspecific interactions. Under the criteria of more than 2 unique peptides, a false discovery rate (FDR) ≤ 1%and a p value &lt;0.05 for the t-test analysis, 147cellular potential proteins were identified (as listed in the table). <b></b><i></i><br>

猪流行性腹泻病毒（Porcine epidemic diarrhea virus, PEDV）是引发猪流行性腹泻（Porcine epidemic diarrhea, PED）的病原，每年给养猪业造成巨额经济损失。截至目前，尚无有效疫苗或药物可用于防控该疫病，因此解析PEDV互作组（PEDV interactome）迫在眉睫。PEDV的核衣壳（N）蛋白在病毒复制过程中发挥关键作用。本研究将PEDV N基因克隆至pEGFP-C1载体，构建重组质粒pEGFP-N。为获取N蛋白的互作组，我们将重组质粒pEGFP-N与空载质粒pEGFP-C1分别转染至293T细胞，随后利用GFP-Trap进行下拉实验以富集细胞内的结合伴侣蛋白。通过无标记质谱（label-free MS）对上述蛋白进行鉴定。结果显示，初始共鉴定并定量到约1200种细胞蛋白，涵盖特异性与非特异性相互作用的蛋白。基于“至少2个独特肽段、错误发现率（false discovery rate, FDR）≤1%、t检验p值<0.05”的筛选标准，最终鉴定得到147种潜在细胞互作蛋白（详见附表）。<b></b><i></i><br>